Amyloid β (Aβ)
Amyloid β is derived from its large precursor protein (APP) by sequential proteolytic cleavages. APP is a single transmembrane polypeptide that is cotranslationally translocated into the endoplasmic recticulum via its signal peptide and then posttranslationally modified throught the secretory pathway. It comprises a heterogeneous group of ubiquitously expressed polypeptides. This heterogeneity arises both from alternative splicing (yielding 3 major isoforms of 695, 751 and 770 residues) as well as from a variety of posttranslational modifications, including the addition of N- and O-linked sugars, sulfation, and phosphorylation. Its acquisition of N- and O-linked sugars occurs rapidly after biosynthesis, and its half-life is relatively brief (45 to 60 minutes). Notwithstanding this heterogeneity, APP is highly conserved in evolution and is expressed in all mammals examined for it; a partial homolog of APP has been found in Drosophilia (APPL). APP is a member of a larger gene family, the amyloid precursor-like proteins (APLPs) which have substantial homology, both within the large ectodomain and the cytoplasmic tail, but are divergent in the Aβ region.
The APP splice forms containing 751 or 770 amino acids are widely expressed in normeuronal cells throughout the body and also occur in neurons. However, neurons express even higher levels of the 695 residue isoform, which occurs at very low abundance in normeuronal cells. The difference between the 751/770-residue and 695-residue forms is the presence in the 751/770-residue isoform of an exon that codes for a 56-amino acid motif that is homologous to the Kunitz-type of serine protease inhibitors (KPI), indicating one potential function of these longer APP isoforms. The KPI-containing isoforms of APP found in human platelets serve as inhibitors of factor Xia, which is a serine protease in the coagulation cascade.
Aβ production is a normal metabolic event Both during and after the trafficking of APP through the secretory pathway, APP can undergo a variety of proteolytic cleavages to release secreted derivatives into vesicle lumens and the extracellular space. The first proteolytic cleavage identified, that made by an activity designated α-secretase, occurs 12 amino acids NH2-terminal to the single transmembrane domain of APP. This processing results in the release of the large soluble ectodomain fragment (α-APPS) into the lumen/extracellular space and retention of an 83-residue COOH-terminal fragment (CTF) in the membrane. Alternatively, some APP molecules not subjected to α-secretase cleavage can be cleaved by an activity designated β-secretase, which principally cuts 16 residues NH2-terminal to the α-cleavage site, generating a slightly smaller ectodomain derivative (β-APPS) and retaining a 99-residue CFT (C99) in the membrane that begins at residue 1 of the Aβ region. The C99 fragment is consequently cleaved in the middle of the transmembrane domain as a result of γ-secretase. Precisely where during its complex intracellular trafficking APP can undergo the α-, β- and γ-secretase remains unknown.
A number of functions have been ascribed to APP holoproteins and/or their major secreted derivative (α-APPs) based on cell culture studies. Soluble α-APPs appear to be capable of acting as an autocrine factor and as a neuroprotective and perhaps neuritotropic factor. In vitro studies indicate that the 751- and 770-residue isoforms (encoding a KPI motif) inhibit serine proteases such as trypsin and chymotrypsin. The secreted APP isoforms can confer cell-cell and cell-substrate adhesive properties in culture. All of these imputed functions have not yet been confirmed in vivo.
In polarized epithelial cells, such as Madin-Darby canine kidney (MDCK) cells, APP is targeted principally to the basolateral membrane, where it can undergo α-secretase cleavage to release α-APPs basolaterally, although a small fraction is targeted and processed apically. In neurons, which are one of the cell types that express the highest levels of APP in the body (particularly APP695), APP can be transported anterogradely in the fast component of axonal transport. APP is present in vesicles in axonal terminals, although not specifically in synaptic vesicles. Cell biological studies demonstrate that APP in the axonal terminals can be transported retrogradely up the axon to the cell body, and some molecules then are fully translocated to the somatodendritic surface. During its retrograde axonal trafficking, some APP molecules can be recycled to the axolemmal surface.
Although it has been assumed that APP axonal terminals might be a principal site for the generation of Aβ, this has not been definitely determined, and APP that recycles in endosomes at various neuronal subsites may be capable of undergoing the sequential β- and γ-secretase cleavages to release the peptide. Although APP is particularly abundantly expressed in neurons and neurons have been shown to secrete substantial amounts of Aβ peptides, other brain cells, which also express APP and release variable amounts of Aβ, including astrocytes, microglia, and endothelial and smooth muscle cells, could contribute to the secreted pool of Aβ that eventually leads to extracellular deposition. Moreover, the fact that (i) virtually all peripheral cells also express APP and generate Aβ and (ii) Aβ is present in plasma raises the possibility that circulating Aβ could cross the blood-brain barrier and contribute to cerebral Aβ accumulation.
Lipid Rafts
It generally is believed that brain lipids are intricately involved in Aβ-related pathogenic pathways. The Aβ peptide is the major proteinaceous component of the amyloid plaques found in the brains of AD patients and is regarded by many as the culprit of the disorder. The amount of extracellular Aβ accrued is critical for the pathobiology of AD and depends on the antagonizing rates of its production/secretion and its clearance. Studies have shown that neurons depend on the interaction between Presenilin 1 (“PS1”) and Cytoplasmic-Linker Protein 170 (“CLIP-170”) to both generate Aβ and to take it up through the lipoprotein receptor related protein (“LRP”) pathway. Further to this requirement, formation of Aβ depends on the assembly of key proteins in lipid rafts (“LRs”). The term “lipid rafts” as used herein refers to membrane microdomains enriched in cholesterol, glycosphingolipids and glucosylphosphatidyl-inositol-(GPI)-tagged proteins implicated in signal transduction, protein trafficking and proteolysis. Within the LRs it is believed that APP is cleaved first by the β-secretase (BACE) to generate the C-terminal intermediate fragment of APP(CFT(C99)), which remains embedded in the membrane. CFT(C99) subsequently is cleaved at a site residing within the lipid bilayer by γ-secretase, a high molecular weight multi-protein complex containing presenilin, (PS1/PS2), nicastrin, PEN-2, and APH-1 or fragments thereof. Aβ finally is released outside the cell where it may: i) start accumulating following oligomerization and exerting toxicity to neurons, or ii) be removed either by mechanisms of endocytosis (involving apolipoprotein-E (apoE) and LRP or Scavenger Receptors) or by degradation by extracellular proteases including insulin-degrading enzyme (IDE) and neprilysin.
Leptin, similarly to methyl-beta-cyclodextrin, reduces beta-secretase activity in neuronal cells. In addition, leptin increases apoE-dependent Aβ uptake in vitro. Like leptin, methyl-beta-cyclodextrin reduces beta-secretase activity in neuronal cells, possibly, but without being limited by theory, by altering the lipid composition of membrane LRs. Like leptin, inhibitors of acetyl CoA carboxylase (e.g. TOFA) and fatty acid synthase (cerulenin) mimic leptin's action, i.e., act as leptin mimics. In contrast, etoxomir, an inhibitor of carnitine palmitoyl transferase-1, is known to increase Aβ production.
Alzheimer's Disease
Alzheimer's disease (also called “AD”, “senile dementia of the Alzheimer Type (SDAT)” or “Alzheimer's”) is a progressive neurodegenerative disorder of the central nervous system (“CNS”). AD is usually diagnosed clinically from the patient history, collateral history from relatives, and clinical observations, based on the presence of characteristic neurological and neuropsychological features.
The pathology of AD includes, but is not limited to, (1) missense mutations in APP, PS1 and PS2 genes; (2) altered proteolysis of Aβ42; (3) progressive accumulation and aggregation of Aβ42 in brain interstitial fluid; (4) deposition of aggregated Aβ42 as diffuse plaques (in association with proteoglycans and other amyloid-promoting substrates); (5) aggregation of Aβ40 onto diffuse Aβ42 plaques and accrual of certain plaque-associated proteins (such as, for example, complement clq, etc.); (6) inflammatory response including (a) microglial activation and cytokine release, (b) astrocytosis and acute phase protein release; (7) progressive neuritic injury within amlyoid plaques and elsewhere in the neuropil; (8) disruption of neuronal metabolic and ionic homeostasis; oxidative injury; (9) altered kinase/phosphatase activities leading to hyperphosphorlyated tau which leads to PHF formation; (10) widespread neuronal/neuritic dysfunction and death in hippocampus and cerebral cortex with progressive neurotransmitter deficits; and (11) dementia. The ultimate effects that may further present in the affected cortical regions include neuritic dystrophy, synaptic loss, shrinkage of neuronal perikarya, and selective neuronal loss.
AD is further characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions. This loss results in gross atrophy of the affected regions, including degeneration in the temporal lobe and parietal lobe, and parts of the frontal cortex and cingulate gyms. Both amyloid plaques (“AP”) and neurofibrillary tangles (“NFT”) are clearly visible after silver staining by microscopy in brains of those afflicted with AD.
Plaques
Amyloid plaques are dense, mostly insoluble deposits of amyloid-beta (“Aβ”) protein and cellular material outside and around neurons. Dystrophic neurites that contain amyloid precursor protein (“APP”) are seen in traumatic brain injury, and “diffuse plaques” can be observed in association with dementia pugilistica, but the appearance of amyloid plaques in AD is unique. The particular appearance of neuritic plaques (i.e., Aβ peptide-containing extracellular lesions surrounded by tau neurofibrillary pathology) is considered specific for AD. It generally is believed that amyloid plaques are not a nonspecific reaction to neurofibrillary pathology because non-AD tauopathies lack amyloid plaques. Further attesting to the specificity of AD-type amyloid plaques is the fact that mutations or duplications of the APP gene as in Down's syndrome produce the specific features of AD, clinically and pathologically. Accordingly, AD involves a specific combination of neuritic amyloid plaques and NFTs where the neuritic plaques seem more specific to the disease, and the NFTs seem more likely to induce neurodegeneration.
Neuritic Plaques
Neuritic plaques comprise roughly spherical extracellular amyloid deposits that are invested by degenerating or dying back nerve cell processes. These abnormal dendrites and axons contain aberrant tau fibrils identical to those seen in NFTs. In a given neuritic plaque, axons from a variety of different sources expressing distinct neurotransmitter signatures may be present. These neurites often are dilated and tortuous and are marked by ultrastructural abnormalities that include enlarged lysosomes, numerous mitochondria, and paired helical filaments, the latter indistinguishable from those that comprise the neurofibrillary tangles. Such plaques also are intimately associated with microglia expressing surface antigens associated with activation, such as CD45 and HLA-DR; they are surrounded by reactive astrocytes displaying abundant glial filaments. The microglia usually are within and adjacent to the central amyloid core of the neuritic plaque, whereas the astrocytes often ring the outside of the plaque, with some of their processes extending centripetally toward the amyloid core. The time that it takes to develop such a neuritic plaque is unknown, but these lesions probably evolve very gradually over substantial period of time, perhaps many months or years. The surrounding neuritis that contributes to any one plaque can emanate from local neurons of diverse neurotransmitter classes. Much of the fibrillar Aβ found in the neuritic plaques is the species ending at amino acid 42 (Aβ42), the slightly longer, more hydrophobic form that is particularly prone to aggregation. However, the Aβ species ending in amino acid 40 (Aβ40), which normally is more abundantly produced by cells than is Aβ42, usually is colocalized with Aβ42 in the plaque. The cross-sectional diameter of neuritic plaques in microscopic brain sections varies widely from 10 μm to greater than 120 μm, and the density and degree of compaction of the amyloid fibrils that comprise the extracellular core also shows great variation among plaques.
Neuritic plaques represent a nidus in which the extracellular amyloid plaque pathology induces intracellular neurofibrillary pathology and apparent structural and functional disruption.
Diffuse Plaques (Preamyloid Deposits)
Many Aβ deposits lack the compacted, fibrillar appearance of the classical neuritic plaques. Studies have indicated that many of the plaques found in limbic and association cortices, and virtually all of those found in brain regions not clearly implicated in the typical symptomatology of AD (for example, thalamus, caudate, putamen, cerebellum), show relatively light, amorphous Aβ immunoreactivity to diagnostic antibodies developed to endogenous Aβ or synthetic Aβ that occurs in a finely granular pattern, without a clearly fibrillar, compacted center. The detection of these plaques in regions that also contain many neuritic plaques led to the hypothesis that these plaques represent precursor lesions of neuritic plaques, and thus are referred to as “diffuse plaques” or “preamyloid deposits.” The Aβ peptides deposited in AD brain principally ends at either Aβ40 or Aβ42. Studies have indicated that peptides that end at Aβ42 are subunits of the material comprising the diffuse plaques, with little or no Aβ40 immunoreactivity, in contrast to the mixed (Aβ42 plus Aβ40) deposits that generally are found in the fibril-rich neuritic plaques.
Neurofibrillary Tangles
Many neurons in the brain regions typically affected in AD (entorrhinal cortex, hippocampus, parahippocampus gyms, amygdale, frontal, temporal, parietal and occipital association cortices, and certain subcortical nuclei projecting to these regions) contain large, nonmembrane-bound bundles of abnormal fibers that occupy much of the perinuclear cytoplasm. Most of these fibers consist of pairs of 10 nm filaments wound into helices (paired helical filaments (PHF)), with a helical period of about 160 nm. Some tangle-bearing neurons also contain skeins of straight, 10 nm to 15 nm filaments interspersed with the PHF. Neurofibrillary tangles (NFTs) are aggregates of the microtubule-associated protein “tau”, which have become hyperphosphorylated and accumulate inside the cells themselves. Tau is relatively abundant in neurons but is present in all nucleated cells and functions physiologically to bind microtubules and to stabilize microtubule assembly for polymerization. The tau gene, comprised of over 100 kilobases and containing 16 exons, contains consensus binding sites for transcription factors such as AP2 and SP1. In the adult brain, alternative splicing of tau nuclear RNA transcribed on exons 2, 3 and 10, results in six tau isoforms, each (i) having either 3 or 4 peptide repeats of 31 or 32 residues in the C terminal region encoded on exon 10, (ii) comprising the microtubule binding domain or (iii) differing in the expression of 0, 1 or 2 inserts encoded on exons 2 and 3. These tau isoforms, as well as their phosphorylation status, change during development such that 3 repeat (“3R”) tau with no inserts is expressed in the fetus and early postnatal infant, while heterogeneous isoforms are expressed in the adult brain. This switch in RNA splicing also corresponds to a reduction in tau phosphorylation.
During neurodegeneration, tau is phosphorylated abnormally at proline directed serine/threonine phosphorylation sites, which can be detected using specific antisera. These serine/threonine (Ser/Thr) phoshorylation sites include Ser-202/Thr-205 (AT8 site), Ser-214 and/or Ser-214, Ser-181, and/or Ser-212 (AT100 site), Thr-231 and/or Ser-235 (TG3 site), and Ser-396/Ser-404 (PHF-1 site). The profile of alternative tau splicing differs among pathological phenotypes, such that tau accumulation in AD is a mixture of 3R and 4R tau, Pick disease tends to be 3R tau, corticobasal degeneration and progressive supranuclear palsy tends to be 4R tau, and so-called agryrophilic grain disease accumulates small inclusions comprised of 3R tau. The general term “tauopathy” encompasses the broad classification of neurodegenerative diseases that accumulate phosphorylated tau.
A variety of kinases have been shown to be capable of phosphorylating tau in vitro at various sites. Nevertheless, it has not become clear whether one or more kinases are principally responsible for initiating the hyperphosphorylation of tau in vivo that leads to its apparent dissociation from microtubules and aggregation into insoluble paired helical filaments.
The correlation between regional distribution of phosphorylated tau and clinical signs suggests a close relationship between tau and AD pathogenesis. The increased tau phosphorylation that accompanies AD may result in separation of tau from the microtubule, possibly aided by other factors (such as, for example, Aβ, oxidative stress, inflammatory mediators), and sequestration of NFTs and neuropil threads. Without being limited by theory, the loss of normal tau function (stabilization and maintenance of microtubules), combined with a toxic gain of function, could compromise axonal transport and contribute to synaptic degeneration. The role of NFT toxicity, however, remains unclear. Studies have indicated that mice models expressing a repressible human tau still developed NFTs, neuronal loss, and behavioral impairments; after tau suppression, the behavioral deficits stabilized, yet NFTs continued to accumulate. In another AD-like model, axonal pathology with accumulation of tau proceeded plaque deposition. NFTs (and presumable “intermediates”) exist within the cytoplasm of viable neurons. Only in advanced disease are large numbers of extracellular NFTs identified.
NFTs are not specific for AD, particularly if a broader definition of NFTs includes different tau isoforms or if one expands the expectation of the morphological characteristics of NFTs. The two classical lesions of AD, neuritic plaques and NFTs, can occur independently of each other. Tangles composed of tau aggregates that are biochemically similar to, or in some cases, indistinguishable from those in AD have been described in more than a dozen less common neurodegenerative diseases, in almost all of which one finds no Aβ deposits and neuritic plaques.
NFTs appear in multiple brain diseases, and may contribute to neurodegeneration in more than one disease state. In addition to AD, NFTs also are found in some frontotemporal dementias, myotonic dystrophy, viral panencephalitis, dementia pugilistica, some prion diseases, and other brain diseases. For many of these disorders, the severity of NFT pathology is less than that observed in end-stage AD. Further, no condition characterized by widespread neocortical NFTs lacks extensive neurodegeneration and clinical dementia. On the other hand, there are many subtypes of chronic brain diseases in which there are extensive neurodegeneration and clinical dementia without NFTs, such as many subtypes of frontotemporal dementias, synucleinopathies, subacute or chronic infarcts, metabolic, demyelinating, developmental, and trinucleotide repeats diseases. Tau protein itself can directly trigger neurodegeneration: many germ line mutations in tau produce clinical dementia with NFTs. These tauopathies are distinct from AD, but common pathways may be involved. NFTs appear in multiple brain diseases, and may contribute to neurodegeneration in more than one disease state.
Dystrophic Cortical Neurites Within and Outside Neuritic Plaques
Many of the dilated and tortuous neurites found within and immediately surrounding amyloid plaques contain PHF that are structurally, biochemically and immunocytochemically indistinguishable from those that comprise the NFTs. In addition, plaques often contain numerous dystrophic neurites that are not immunoreactive for PHF tau. Tau-positive dystrophic neurites also are present in a more widespread distribution in the cortical neuropil outside of the neuritic plaques. The prevalence and density of dystrophic cortical neurites that contain altered forms of tau varies substantially among AD patients. Studies have shown that AD patients particularly rich in NFTs also are those that show widespread tau-immunoreactive dystrophic cortical neurites. Some of the intraplaque and extraplaque dystrophic neurites are immunoreactive for phosphorylated forms of the neurofilament subunit proteins; thus phosphorylated forms of the neurofilament subunit proteins can coexist with phosphotau reactivity. These observations suggest that there may be several substrates for the altered kinase and phosphatase activities that occur in tangle-bearing neurons and dystrophic neurites.
Amyloid Microangiopathy
Aβ originally was isolated from amyloid-laden meningeal arterioles and venules that often are found just outside of the brains of patients with AD or Down's syndrome. Similarly, small arterioles, venules, and capillaries within cerebral cortex also frequently bear amyloid deposits. This microvascular angiopathy is characterized at the ultrastructural level by amyloid fibrils found in the abluminal basement membrane of the vessels, sometimes with apparent extension of the fibrils into the surrounding perivascular neuropil (dyshorric angiopathy). The Aβ peptides that occur as filaments in the microvessel basement membranes appear, on the basis of immunoreactivity, to be principally Aβ40 species, although evidence has been presented that the initially deposited species in vessels destined to develop amyloid angiopathy may be Aβ42. The extent of amyloid angiopathy varies widely among AD brains that have relatively similar burdens of parenchmyal Aβ. The contribution of this microvascular amyloidosis to the cortical dysfunction that occurs in AD and the mechanism by which amyloid alters microvascular function remains unknown. Amyloid-bearing vessels composed of Aβ deposits essentially indistinguishable from those of AD can occur in the virtual absence of parenchymal Aβ deposits in the brains of elderly subjects without AD. Such amyloid-bearing vessels in this condition (congophilic amyloid angiopathy (CAA)) as well as those in AD can occasionally rupture, leading to one or multiple cerebral hemorrhages. Nevertheless, the large majority of AD patients do not experience cerebral hemorrhages, despite the presence of some or many microvascular amyloid deposits.
The Principal Underlying Cause of Alzheimer's Disease Remains Unknown
The principal underlying cause of AD remains unknown. Disagreements persist as to whether Aβ peptide-rich plaques or NFTs are the principal neuordegenerative element and whether they are etiologically related. There is a high degree of disparity among research efforts to address whether there are earlier biochemical events that ultimately lead to the characteristic pathology. It generally is believed that soluble Aβ oligomers, prior to plaque buildup, exert neurotoxic effects leading to neurodegeneration, synaptic loss and dementia. Further, increased Aβ levels may result from abnormal lipid accumulation, thereby producing altered membrane fluidity and lipid raft composition. However, for sporadic AD, representing the overwhelming majority of AD cases, there still is no convincing evidence for a particular cause that triggers the Aβ cascade.
Leptin
Leptin is a helical protein, secreted by adipose tissue, which acts on a receptor site in the ventromedial nucleus of the hypothalamus to curb appetite and increase energy expenditure as body fat stores increase. Leptin levels are 40% higher in women, and show a further 50% rise just before menarche, later returning to baseline levels. Leptin levels are lowered by fasting and increased by inflammation.
Ablation of leptin or of leptin signaling is sufficient to cause obesity as exemplified by leptin-deficient obese, hyperinsulinemic mice having the genotype ob/ob; diabetic mice with a mutation in the leptin receptor gene having the genotype db/db, which produce but are non-responsive to leptin; rats of the genotype fa/fa, which have the “fatty” obesity gene, which is a mutated leptin receptor; and in a few rare genetic cases (Schwartz et al., Nature. 404: 661-71 (2000)). Laboratory mice having mutations on the ob gene, which encodes leptin, become morbidly obese, diabetic, and infertile; administration of leptin to these mice improves glucose tolerance, increases physical activity, reduces body weight by 30%, and restores fertility. Mice with mutations of the db gene, which encodes the leptin receptor, also become obese and diabetic but do not improve with administration of leptin. Human genes encoding both leptin and the leptin receptor site have been identified. Although mutations in both the leptin and leptin receptor genes have been found in a small number of morbidly obese human subjects with abnormal eating behavior, the majority of obese persons do not show such mutations, and have normal or elevated circulating levels of leptin. An immune deficiency seen in starvation may result from diminished leptin secretion. Mice lacking the gene for leptin or its receptor show impairment of T-cell function, and, in laboratory studies, leptin has induced a proliferative response in human CD4 lymphocytes.
Leptin binding to its functional receptor recruits Janus tyrosine kinases and activates the receptor, which then serves as a docking site for cytoplasmic adaptors such as STAT (Baumann, H., et al. Proc. Natl. Acad. Sci. USA 93:8374 1996)). According to the general model for JAK/STAT activation, STAT proteins initially are present in inactive forms in the cytoplasm. Following ligand stimulation and receptor dimerization, the JAK/STAT pathway is activated by activation of receptor-bound JAK kinases. These JAK kinases subsequently phosphorylate the receptor at tyrosine residues, which recruits STATs to the receptor. STATs then are phosphorylated to form phosphoSTATs, dimerized, and translocated to the nucleus, where the phosphoSTAT dimers bind to specific sequences in the promoter regions of their target genes, and stimulate the transcription of these genes (Schindler et al., Ann. Rev. Biochem. 64: 621-51 (1995)), including negative regulators, such as the suppressor of cytokine signaling 3 (Bjorbaek, C., K. et al. J. Biol. Chem. 274:30059 (1999)) and the protein tyrosine phosphatase 1B (Cheng, A. N. et al. Dev. Cell 2:497 (2002), Schwartz et al., Nature, 404:661-71 (2000), Louis A. Tartaglia, J. Biol. Chem. Minireview, 272:6093-6096 (March 1997)).
In addition to the JAK-2-STAT-3 pathway, other pathways also are involved in mediating leptin's effect in the brain and on the immune cells. For example, the mitogen-activated protein kinase (MAPK) pathways, the insulin receptor substrate 1 (IRS1) pathway, and the phosphatidylinositol 3′-kinase (PI3′K) pathway (Martin-Romero, C., V. Sanchez-Margalet. Cell. Immunol. 212:83 (2001)) also mediate leptin's action (Sanchez-Margalet, V., C. Martin-Romero, Cell. Immunol. 211:30 (2001)).
Leptin also may have a physiologic role as a liporegulatory hormone responsible for maintaining intracellular homeostasis in the face of wide variations in caloric intake (Unger R H.2003. Annu Rev Physiol. 65:333-47). This is achieved by directly stimulating lipolysis, (meaning fat breakdown), and inhibiting lipogenesis (meaning fat synthesis) (Lee Y, et al., J. Biol. Chem. 276(8):5629-35 (2001)). Leptin also can improve insulin resistance and hyperglycemia by a mechanism not completely understood (Toyoshima et al., Endocrinology 146: 4024-35 (2005)), despite insulin's ability to stimulate lipogenesis (Kersten, EMBO Reports 2(4): 282-286 (2001). This aspect of leptin's physiological role is important, because insulin and Aβ share a mechanism for their clearance, namely degradation by insulin degrading enzyme (IDE).
Leptin also controls insulin sensitivity. Within the central nervous system (CNS), leptin crosses the blood brain barrier to bind specific receptors in the brain to mediate food intake, body weight and energy expenditure. In general, (i) leptin circulates at levels proportional to body fat; (ii) leptin enters the CNS in proportion to its plasma concentration; (iii) leptin receptors are found in brain neurons involved in regulating energy intake and expenditure; and (iv) leptin controls food intake and energy expenditure by acting on receptors in the mediobasal hypothalmus.
It generally is believed that leptin inhibits the activity of neurons that contain neuropeptide Y (NPY) and agouti-related peptide (AgRP), and increases the activity of neurons expressing α-melanocyte-stimulating hormone (α-MSH). The NPY neurons are a key element in the regulation of appetite; small doses of NPY injected into the brains of experimental animals stimulates feeding, while selective destruction of the NPY neurons in mice causes them to become anorexic. Conversely, α-MSH is an important mediator of satiety, and differences in the gene for the receptor at which α-MSH acts in the brain are linked to obesity in humans.
AMP-Activated Protein Kinase (AMPK)
AMP-activated protein kinase (AMPK) is a phylogenetically conserved serine/threonine protein kinase that exists as a heterotrimeric complex consisting of a catalytic subunit α and two regulatory β and γ subunits. The conventional serine/threonine activity of AMPK is supported by its α subunit, which is characterized by the presence (in the activation loop) of a threonine residue (Thr172) whose phosphorylation is required for activation. The C-terminal region of α subunit is required for association with the other two β and γ subunits. The β subunit contains a C-terminal region required for the association with α and γ subunits and a central region that allowed AMPK complex to bind glycogen. The γ subunit contains four tandem repeats known as cystathionine β-synthase (“CBS”) motifs which bind, together, two molecules of AMP or ATP in a mutally exclusive manner. Binding of AMP (on γ subunit) activates AMPK via a complex mechanism involving direct allosteric activation and phosphorylation of α subunit on Thr172 by upstream kinases such as the protein kinase LKB1 (a tumor suppressor whose germline mutations in humans are the cause of Peutz-Jeghers syndrome), the CaMKKIIβ (calmodulin-dependent protein kinase kinase IIβ) and potentially TAK1 (mammalian transforming growth factor β-activated kinase).
Homologues of all three subunits have been identified in mammals, fruitfly (Drosophila melanogaster), worm (Caenorhabditis elegans), yeast (Saccharomyces cerevisiae), plants (Arabidopsis thaliana) and the primitive protozoan Giardia lamblia, with a high degree of conservation that suggests that this protein evolved at least a billion years ago to regulate a wide spectrum of actions on metabolic homeostasis. In mammals, two to three isoforms of each subunit (α1, β2, β1, β2, γ1, γ2, γ3) encoded by different genes are known giving rise to a large variety of heterotrimeric combinations, with splice variants (for the γ2 and γ3 genes) adding to the diversity. Furthermore, differences in the tissue distribution of the expression patterns of catalytic and regulatory isoforms have been reported.
Studies of isoform composition of AMPK complexes in human skeletal muscle found that only 3 of the 12 theoretically possible AMPK complexes were present (α2β2γ1>>α2β2γ3=α1β2γ1) and were activated differently depending on exercise intensity and duration. Moreover, specificity of each catalytic isoform has been shown for its preferentially upstream kinase in both skeletal muscle and heart; indeed, in LKB1−/− mice, ischemia in the heart and contraction in skeletal muscle were not able to activate AMPKα2 subunit, whereas AMPKα1 activation was only slightly affected. Expression of the γ3 subunit appeared highly specific to glycolytic skeletal muscle whereas γ1 and γ2 showed broad tissue distributions. In skeletal muscle, the β2 subunits also is highly expressed but the β1 subunit predominates in the liver. AMPKα1 and α2-containing complexes account each for about half of total AMPK activity in liver. In adipose tissue, AMPK complexes containing the al catalytic subunit are mainly expressed whereas, in skeletal and cardiac muscles, AMPK complexes containing the α2 catalytic subunit are predominant.
In addition to differences in tissue distribution, it generally is believed that distribution of AMPK complexes also is regulated at the intracellular level. AMPKα2-containing complexes have been found in both the nucleus and the cytoplasm; this raises the possibility of the direct phosphorylation of co-activators and transcription factors. In contrast, AMPKα1-containing complexes are predominantly localized in the cytoplasm but also have been observed at the plasma membrane in airway epithelial cells and cartoid body cells. Although the functional significance of different AMPK isoform combination, as well as the function of each heterotrimeric AMPK complex in relation with its particular sensitivity to AMP and ATP, subcellular localization and/or specific targets remains unclear; it has been hypothesized that regulation of exercise-induced glucose transport in human skeletal muscle could be associated with α2β1 rather than α2β2γ3 heterotrimeric complex activation. It has been further suggested that isoform combination also may determine subcellular targeting of AMPK and hence targeting substrates. Studies have shown that the post-translational modification of the β1 subunit may target AMPK complexes to the plasma membrane. In addition, it was found that plectin, a cytoskeleton linker protein which has been shown to bind the γ1 subunit, affects the subunit composition of AMPK complexes in differentiated myotubes. Thus, the selective expression of a particular AMPK complex could determine a specialized cellular and systemic response to different metabolic stresses.
There have been very few studies of the metabolic pathways triggered by leptin in AD pathobiology, and the role of 5-adenosine monophosphate protein kinase (AMPK) in AD pathobiology remains unclear.
The described invention, which provides methods related to treating progressive cognitive dysfunction resulting from accumulations of NFTs or Aβ, also provides methods and compositions for treating AD utilizing leptin and its role in the regulation of two major AD pathways via distinct AMPK-dependent mechanisms in neuronal cells. This bimodal action of leptin, and potentially of AMPK activators, provides a novel therapeutic approach to AD treatment. Currently approved therapies fail to target any of the AMPK-related facets of the disease and provide only symptomatic relief. Further, current investigational drugs address, at most, only one AMPK-related aspect.